Proteins are important constituents of foods. Proteins are a major source of energy, and proteins contain essential amino-acids, such as lysine, tryptophan, methionine, leucine, isoleucine and valine, which are essential to human health. Proteins are the major structural components of many natural foods, and a particular protein in a food can determine the food structure or texture, e.g., tenderness of meat or fish products. Some individual proteins are used as food ingredients because they can improve appearance, texture or stability. Such individual proteins are employed as gelling agents, emulsifiers, foaming agents, and thickeners.
There is an increasing requirement for protein production in academic and industrial settings for a variety of applications like drug discovery, biopharmaceutical production, and the food industry. Currently, large-scale production strategies are capable of providing cell cultures or fermentation titres containing 25-50 wt-% of the desired protein, and the remaining 50-75 percent impurities. Current applications require that essentially all impurities be removed from a highly purified protein product, and that the highly purified protein product can be produced in commercial quantities.
Heme protein refers to a metallo protein which contains a reduced iron atom, Fe2+ in the center of a complex hydrophobic structure. Heme proteins play a role in critical physiological functions because the iron atom in heme protein make heme proteins responsive to molecules which can bind to divalent iron, such as oxygen, nitric oxide, carbon monoxide, and hydrogen sulfide. Hemoglobin and myoglobin are types of heme proteins which are essential for storing and transporting oxygen in mammals. Hemoglobin is also found in the root nodules of some plants. Leghemoglobin (legHb) occurs in the root nodules of leguminous plants, where it facilitates the diffusion of oxygen to symbiotic bacteriods in order to promote nitrogen fixation.
Plant proteins, such as Ribulose-1,5-bisphosphate carboxylase oxygenase, most commonly known by the shorter name RuBisCO, is an plant enzyme involved in the Calvin cycle that catalyzes the first major step of carbon fixation, a process by which atmospheric carbon dioxide is made available to organisms in the form of energy-rich molecules such as glucose. RuBisCO is very important in terms of biological impact, because it catalyzes the primary chemical reaction by which inorganic carbon permanently enters the biosphere. RuBisCO is also the most abundant protein in leaves, and is considered to be the most abundant protein on Earth.
Cellulose is the main component of higher plant cell walls and one of the most abundant organic compounds on earth. It can be derived from a number of sources using a number of techniques that are considered synthetic, and some that might be considered non-synthetic (natural). It is available in many forms for different functional purposes in food products. The three main forms of cellulose that have been considered for various uses are powdered cellulose, regenerated cellulose casing, and microcrystalline cellulose. Microcrystalline cellulose is purified, partially depolymerized cellulose. It is a fine, white, odorless crystalline powder which is insoluble in water, insoluble in dilute acids, insoluble in most organic solvents, and also insoluble in dilute sodium hydroxide solutions. Microcrystalline cellulose is primarily used in food to stabilize and improve the body, texture, and stability of food products.
Ion exchangers which have been used in separating whey proteins from whey protein containing solutions include both cation exchangers, particularly of the SP or SE (sulphonate) or CM (carboxymethyl) type, and anion exchangers, particularly of the QA (quaternary amino) or DEAE (diethylaminoethyl) type. In terms of the exchanger matrix itself, many insoluble matrices have been used, including cellulose, cross-linked dextran, cross-linked agarose, synthetic hydophilic polymers and inorganic materials coated with hydrophilic polymers. One matrix that has proved to be particularly useful in large scale separation and purification of whey proteins is regenerated cellulose which has been hydroxyalkylated and cross-linked. Ion exchangers prepared on this matrix are resistant to attrition, have high protein capacity, high flow properties and are available at relatively low cost.
Preparation of quaternized celluloses is known in the art, for example from U.S. Pat. No. 3,472,840 assigned to Union Carbide Corporation, which discloses cellulose derivatives, particularly cellulose ethers containing quaternary ammonium groups which are used in the many fields in which cellulose ethers cannot be employed.
U.S. Pat. No. 6,911,483 to Ayers et al. discloses an anion exchanger comprising a water insoluble, hydrophilic, water swellable, hydroxy(C2-C4 alkylated and cross-linked regenerated cellulose, derivatized with quaternary amino (QA) groups. Ayers et al. discloses and claims that such anion exchangers have a level of substitution of the QA groups of 1.4 milliequivalents per dry gram of anion exchanger (meq/g) or greater. Anion exchangers are useful for separating proteins from protein-containing solutions, and particularly for adsorbing whey proteins from whey protein-containing solutions. Ayers et al. further differentiates such anion exchanger compositions based on regenerated cellulose from materials produced from microcrystalline cellulose with reference to Antal et al. Antal et. al. (Carbohydrate Polymers 19, 167-169, 1992) describe the optimization of the reaction of microcrystalline cellulose with the alkylating agents CHPTAC-(chloro-2-hydroxypropyl)trimethylammonium chloride) and 1,3-bis(3-chloro-2-hydroxy-propyl)imidazolium hydrogen sulfate in alkaline medium. Ayers et al. states that the maximum substitution level they [Antal et al.] were able to obtain with CHPTAC was 0.94 meq/g (mmol/g, millimoles per gram)), although the second reagent gave a product with 1.56 meq/g. No protein capacities are given and it is likely that the latter reagent, being bifunctional, would have introduced extensive crosslinking into the cellulose to the detriment of protein capacity. Furthermore, Ayers et al. states that microcrystalline cellulose is not a suitable matrix for repeated use on a large industrial scale.
Fibrous cellulose has been derivatized with quaternary ammonium groups to a high degree of substitution, DS of at least 0.5 (>2 meq/g), using a very large excess of alkylating reagent containing quaternary ammonium groups. The cellulose is either not crosslinked (1998 U.S. Pat. No. 5,731,259) or crosslinked (1998 U.S. Pat. No. 5,780,616). Preferably the alkylating reagent is used in 20:1 to 40:1 mole ratio of reagent to anhydroglucose units of cellulose. In the case of GTAC this amounts to 186-372 g of reagent per 10 g of cellulose used either in 5-8 repeated reactions or one large addition of the solid reagent with 30 mL of water. The products, described at one point as a jelly mass, are useful as superabsorbents for water and saline solutions in the field of hygenic-sanitary products such as diapers for babies. They are designed to be used once and then disposed of and are not at all suitable for repeated use day after day in a reactor or column bed where physical robustness against attrition, long life and high flow-through rates are required for anion exchangers processing protein solutions.
Processes are sought for the large scale separation and continuous purification of proteins, such as heme proteins and plant proteins, from mixtures of protein-containing solutions such as animal and vegetable derived protein extracts.